Book - The Hormones in Human Reproduction (1942) 5
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Chapter V. A Hormone for Gestation
- "In general estrogen is the hormone of the woman, it assures the development of the genital and mammary apparatus ; progesterone is the hormone of the mother, it is indispensable for reproduction* - Robert Courrier, Biologie des Hormones Sexuelles Femelles, 1937 (translation).
If this were a detective story, not merely a story of detection, the reader would at this point be directed to turn back to pages 41-44, in which (he would solemnly be informed) he will find all the clues necessary to solve the great Corpus Luteum Mystery. Having reread those pages and having inspected the photographs of the corpus luteum, Plate IX, he will be in possession of all the information that enabled Louis-Auguste Prenant of Nancy in 1898 to suggest that the corpus luteum, subject of so much previous conjecture and so little fact, is actually an organ of internal secretion. This guess, I admit, required sharp wits. Today every reader who has studied biology ought to get the same idea when he sees this small but vivid organ, walled off from the rest of the ovary by its fibrous capsule, drained by no secretory duct but obviously equipped with a rich network of blood vessels, and composed of large and characteristic cells resembling those of the adrenal gland. Seen under the microscope as pictured in Plate IX, C, this arrangement fairly shouts "I am a gland of internal secretion" ; to recognize that fact forty-four years ago was a real feat of scientific detection.
But even in novels of crime the sleuth's clever guess must be followed by careful accumulation of evidence for the jury; still more is this the case when the object of detection is the function of an important gland and the seeker's reward a valuable addition to scientific knowledge and human welfare.
I do not pretend to write this chapter in cool detachment. Its theme-word progesterone has for me connotations that will never be found in the dictionary. In the first place I invented the word myself, as far at least as the letter "t," as will be explained hereafter. In the second place, it recalls memories of bafflement, comedy, hard work, and modest success. Can I forget the time I went racing up the steps of the laboratory in Rochester, carrying a glass syringe that contained the world's entire supply of crude progesterone, stumbled and fell and lost it all.? Or the day Willard Allen showed me his first glittering crystals of the hormone, chemically pure at last?
In the third place, I am writing largely about the work of personal friends. Prenant and Born I did not know, for chronological reasons ; but Paul Bouin received me in his laboratory at Strasbourg many times in the summer of 1924), looking like a Frenchman out of a storybook, writing and teaching like the grand scholar and gentleman he is. The story of Born's legacy I heard from Ludwig Fraenkel himself, to whom in Montevideo may this book carry a special greeting. Dispossessed of his famous clinic in Breslau, driven from his country, he can never be exiled from a world that honors great minds and great hearts wherever they are. Karl Slotta and Eric Fels, when at last I met them in their South American homes, proved no less distinguished in hospitality than in chemistry. Adolf Butenandt of Berlin sat happily at my own fireside and dinner table in 1935, and there he will be welcome again now that his country's guns are silenced.
Nor can I possibly write with detachment of Willard Allen's work, which at first I shared and afterward watched with affectionate admiration. The American pioneers in this work, Leo Loeb and Robert T. Frank, early honored me with their acquaintance and good will. Likewise the names of our Wisconsin fellow-workers, Frederick Hisaw (now at Harvard), Harry Fevold, Charles Weichert, Samuel Leonard, Roland Meyer (the latter for three years also in my laboratory at Rochester) are written not only in the formal list of investigators to be mentioned here, but also in memory's record of friendly rivalry and mutual enthusiasm. Without apology, then, let these personal feelings color (for so they must) the narrative of research.
The collection of evidence begins with a scene poignant enough, indeed, for a novel. In 1900 the great embryologist of Breslau, Gustav Born, lay dying. Scientist to the last, his mind was full of a hypothesis he knew he could not live to test and which he could not bear to leave untried. To his bedside, therefore, he summoned one of his former students, the rising young gynecologist Ludwig Fraenkel. To him Born imparted his thought that the corpus luteum is indeed an organ of internal secretion, and moreover that its function must be concerned with the protection of the early embryo. This guess about its specific function, like Prenant's about its general nature, was brilliant and novel in its day, even though to us in retrospect when we consider that the corpus luteum is present only when the egg is available for development, such a function seems probable indeed. So it seemed then to Fraenkel, whose task it was to devise the experiments by which Born's conjecture could be put to rigorous test.
He knew that in the rabbit the embryos become implanted in the uterus on the 8th day after mating. They spend 3 days in the oviduct and then 4 days more as free blastocysts in the uterus, before they become attached. To test the function of the corpora lutea, Fraenkel planned to interfere with the natural course of events by removing them while the embryos were still unattached. The simplest way of removing the corpora lutea is of course to remove both ovaries, by surgical operation under an anesthetic. Since this might, for all he knew, remove some other useful or necessary factor, Fraenkel tried also cutting out the corpora lutea alone, or burning them out with a fine cautery, of course always under an anesthetic. This operation is more difficult than simple removal of the ovaries. The rabbit sheds many eggs at a time, up to 10 or even 12, and a corresponding number of corpora lutea have to be searched for and removed at the operation.
As already explained on p. 68, rabbits are peculiar in that the ovarian follicles ripen only after mating, not spontaneously at more or less regular intervals, as in other animals. This is a great advantage for the present purpose, for it means that we can time our experiments at will. Fraenkel simply mated his females to buck rabbits of known fertility. He knew they would ovulate next day and that while the eggs were being fertilized and beginning to develop, the ruptured follicles would be transformed into corpora lutea. Sometime during the next 6 days he intervened surgically and destroyed the corpora lutea. Then he simply waited to see what happened. If loss of the corpora did nothing, he could expect that after 3 weeks the rabbit would show signs of pregnancy and about the 33rd day, as is the rule in this species, she would give birth to her litter of young. Actually, when the experiment was performed as described, no pregnancy ensued. Something had interfered with the embryos. Fraenkel checked this result by careful control experiments; he removed only one ovary, or cut into both ovaries without removing the corpora lutea. Thus he had experiments in which there was no endocrine loss, but just as much upset and damage as if the corpora lutea had been removed. The results were decisive. If the corpora lutea were not completely removed, the pregnancy went on. If they were removed, the pregnancy failed. In many cases he did not wait for the time of birth, but autopsied the rabbit 3 or 4 weeks after operation, always finding that the embryos had disappeared from the uterus. He did not learn what had actually happened to them, nor when the blow fell. He only knew they could not survive the loss of the corpora lutea.
These results were presented to the German Gynecological Society in 1903, but they met a good deal of criticism and disbelief. Some of the experiments were for technical reasons not perfectly convincing, although we know now their outcome was correct. Fraenkel returned to his laboratory and after seven years was able to present in 1910 completely acceptable results.
At about the same time as these later experiments, and apparently without knowledge of Fraenkel's work, the French histologist Bouin and his colleague Ancel had under way experiments (published in 1910) which demonstrated another aspect of the activity of the corpus luteum. They found that during early pregnancy the lining of the uterus undergoes a remarkable change. This is shown in Plate XVII, B, which reproduces one of Ancel and Bouin's actual figures. The tubular glands of the uterus begin to grow longer, to secrete fluid and therefore to become dilated. Their cells multiply so fast that there is no longer enough room for them in the simple tubular wall, and the glands begin to become folded or pleated. The folds of the endometrium are deeply pervaded by these glands ; and finally in a section of the uterus one sees a beautiful lace-like pattern (Plate XVII, B, right) representing the cross-section of this gland-filled tissue.
This change in the condition of the lining of the uterus, described by Ancel and Bouin, gave the key to the discovery of the corpus luteum hormone. We shall have to mention it again and again. We can therefore save words by using the technical name of this change, i.e. progestational proliferation, that is to say "growth and change which favors gestation." Obviously, as the picture shows, it is growth and change; that it favors gestation will be proved as we go along.
Anticipating our story again, so that we may be perfectly clear about this important matter, let it be said that progestational proliferation does not occur in the rabbit only, but in all mammals, in the first days of pregnancy and also without pregnancy, whenever a recent corpus luteum is present in the ovary. This means that in animals with regular cyclic ovulation, for example the guinea pig, dog, cat, pig, human, and indeed most mammals, progestational proliferation occurs in each cycle. It is not exactly alike in all animals ; it is very elaborate in some, such as the rabbit and the primates (monkeys, apes, and humans) but in others, e.g. rat, mouse, and guinea pig, it is relatively slight. Plate XVII shows it in three kinds of animals.
Ancel and Bouin guessed that this progestational proliferation is caused by the corpus luteum, and they took steps to prove it by an ingenious plan. They mated their female rabbits, not to fertile bucks, but to males rendered infertile by tying off their seminal ducts. Such rabbits ovulated and formed corpora lutea but, of course, did not become pregnant. The only respect in which they were different after mating was that their ovaries now contained corpora lutea. Since their uteri developed typical progestational proliferation, the corpora lutea must have been responsible. The two experimenters then repeated their experiment of mating their rabbits to infertile males, but within a day or two they removed the ovaries or cut out the corpora lutea. Progestational proliferation did not occur. Obviously the corpus luteum controls the condition of the uterus and determines the occurrence of progestational proliferation.
Plate XVII. Preparation of the uterus for implantation of the embryo (progestational proliferation) in human, rabbit, and pig. In each case the left-hand figure shows the interval stage, the right-hand figure shows the effect of the corpus luteum hormone. A^ this process in the human uterus, from the first description by Hitschmann and Adler, 1908. Magnified about 15 times. B, the first pictures of progestational proliferation of the rabbit's uterus, by Bouin and Ancel, 1910. Magnified about 10 times. C, the same change in the uterus of the sow, from preparations by the author. The left-hand figure represents the day before ovulation, the right-hand section was taken 8 days after ovulation. Magnified 10 times.
The American investigator Leo Loeb had indeed already shown (1909), in another way, that the corpus luteum controls the state of the uterus. In the guinea pig the embryo settles deeply into the uterine lining (endometrium) as it does in the human species, and the maternal tissue responds by active growth at the site of attachment, so that the placenta contains a great mass of maternal cells. Loeb showed that the maternal response is caused to occur by the irritation, so to speak, produced by the embryo as it settles into the lining of the uterus. In his experiments he imitated this irritative action, in nonpregnant animals, by putting into the uterus, not embryos, but bits of foreign material, such as tiny pieces of glass or collodion. In his simplest trials he ran a silk thread through the uterus and tied it in place, or merely inserted a needle into the uterus and scratched the endometrium. At the points of irritation, the interior of the uterus promptly developed within a few days, little tumors made up of cells closely resembling, under the microscope, the maternal part of the placenta.
Plate XVIII. Action of progesterone, the hormone of the corpus luteum. Af normal litter of embryos of rabbit in uterus, 5 days after mating. B, dead and degenerating embryos (same age as those in ^) in uterus of rabbit whose ovaries were removed the day after mating. Magnified 20 times. C, section of uterus at time of ovulation. D, uterus of rabbit castrated just after ovulation and given injections of progesterone for 5 days. Magnified 7 times. E, at (1) section of uterus of infant rabbit 8 weeks old; at (2) same after 5 days' treatment with estrogenic hormone; at (3) same after 5 more days' treatment with progesterone. All magnified 7 times. F, two litters of rabbit embryos 6 days old, from mothers deprived of their ovaries one day after mating but injected with progesterone daily. Magnified 71/2 times. Preparations by author. Courtesy American Journal of Physiology.
Now we come to the point of all this. Loeb discovered that he could get his tumors only during a limited part of each cycle, when the corpora lutea are present. If he tried at other times, or if he tried it at the right time but took away the corpora lutea, then he got no placenta-like tumors. In short, when the corpora lutea are present, the uterus is in a special state in which it can respond to the need of the embryos for maternal protection.
Reviewing the story, we see that Fraenkel demonstrated that the implantation of the embryos depends upon the corpus luteum, while Ancel and Bouin had shown by one experiment, and Loeb by another, that the functional state of the uterus depends upon this same gland. We cannot escape the conclusion that these two facts are connected; in other words, that the corpus luteum fosters the embryos by setting up progestational proliferation. No doubt this would have been proved very promptly had not the World War of 1914-1918 interfered with such investigation.
It fell to my own lot (in 1928) to conduct the experiments which tied together the discoveries of Fraenkel and of Bouin and Ancel, by showing exactly what happens, and when, to embryos deprived of the support normally afforded them by the corpus luteum. In the first of my experiments I mated seven female rabbits to fertile males. Fourteen to eighteen hours thereafter the ovaries were removed by surgical operation under complete anesthesia. At this time, as we know from previous studies by embryologists, the eggs were in the 2-cell stage and were in the oviducts. Five, six, or seven days thereafter the animals were killed and examined. Progestational proliferation had of course not occurred, because the corpora lutea had been removed. When the embryos were recovered, by washing them out of the uterus, it was found that they had died in utero. From their measurements and stage of development, as compared with normal embryos (Plate XVIII, B, A), it could be ascertained that they had ceased to grow on the fourth day, i.e. as soon as they had entered the uterus. These embryos died because the uterus was unprepared to receive them.
A control experiment was done with seven more rabbits.
In these everything was done as before, except the ovaries were not removed. Parts of them were removed, or they were cut in two, leaving their blood supply intact; in short, as much interference and damage was produced as in the first group, but in each case several corpora lutea were left in place. In these rabbits, progestational proliferation of the uterus occurred normally; and in six of the seven, normal embryos were found when the rabbits were killed for study on the 5th to the 8th day.
To sharpen the results, and pin them directly to the corpus luteum, I was luckily able to find seven rabbits in which, when I explored them, the corpora lutea were found to be grouped all together in one end of an ovary. When this chanced to be the case, in either the right or the left ovary, I could take out one ovary entirely and all the grouped corpora lutea from the other, still leaving a large amount of ovarian tissue. In all these, progestational proliferation failed to occur and the embryos died.
We have proved two points. First, we have shown that successful care of the embryos in utero depends upon a chain of events. The corpora lutea prepare the uterus, the uterus then cares for the embryos. The reason, unknown to Fraenkel, that his embryos died was that he had prevented Ancel and Bouin's progestational proliferation, by removing the corpora lutea.
Second, we have found that the corpora lutea are necessary not only for implantation (as indicated by Loeb's experiment), but also still earlier, for the nutrition and protection of the embryos during the time when they are lying free in the uterus. How they do this is another question which will be considered later; obviously it is a matter of chemical substances secreted by the glands of the uterine lining under the influence of the corpus luteum (Appendix II, note 4).
If the corpus luteum can do these remarkable things by hormone action, we ought to be able to get the hormone out of the gland and purify it ; but how are we to know when we have it and how much we have? This question is answered by our experiments on rabbits, just cited. All we need to do is to mate a rabbit, remove the ovaries next day, and then administer our extract to see whether it will cause progestational proliferation, and if so, how much extract is required.
The Hormone of the Corpus Luteum
From this stage on I was fortunate indeed in having the collaboration of Willard M. Allen, then a medical student equipped with an excellent knowledge of organic chemistry. We began, of course, in the dark. All we knew for certain was that we had to extract something; we did not know what it was or what its chemical properties might be. We had two clues. In the first place, practically all the known important chemical substances in the animal body can be dissolved and therefore extracted by either water or alcohol, provided they are protected from breaking down, spoiling, or being digested in the process. (Incidentally, how can you protect a substance from spoiling if you do not know what it is?) In the second place, we had a hint from the work of Edmund Herrmann, mentioned in Chapter IV, p. 82. Some of his photographs, published in 1915 in the report of his work on the ovarian hormone (i.e. estrogen) showed that without realizing it he had produced progestational proliferation with some of his extracts. From his report we knew that whatever he had in his extracts must be soluble in alcohol. Willard Allen and I began therefore by collecting corpora lutea of sows' ovaries from the slaughterhouse. We minced them up in a meat chopper and extracted them with hot alcohol. Very luckily for us, Walter R. Bloor, then professor of biochemistry at Rochester, is a great expert on animal fats and allied substances. We built our extractors from his design and sought his advice on many details of the chemical manipulation. After various tribulations, one of which I shall narrate below (page 118), we found (1929) that we could obtain a crude oily extract, looking like a poor grade of automobile grease, which when injected into experimental rabbits was a perfect substitute for their own ovaries in tests such as described above. After 5 days' injection, progestational proliferation was complete. This is well illustrated by comparison of Figures C and D on Plate XVIII, which show sections of the uterus of a castrated rabbit before and after treatment.
In some of our experiments, in which a large dose was used, we even improved upon nature by producing more extensive progestational proliferation than normally occurs. What was even more striking, the embryos at 6 days were just as well off as if their mother's own ovaries had seen to their welfare. Plate XVIII, F shows two such litters of embryos 6 days old, in rabbits castrated more than 5 days before. Evidently our crude oily extract contained the long-sought hormone.
At the University of Wisconsin, at the same time, F. L. Hisaw and his associates, Meyer, Weichert and Fevold, were also engaged in the extraction of sows' ovaries in the search for an active substance. Their reason for looking for a hormone was, however, curiously different. In the pregnant guinea pig, as the time of birth approaches, the two pelvic bones, right and left, become separated where they join in front ; that is, on the belly side of the animal. The bony junction in fact almost melts away, in order to allow birth of the infant guinea pigs, which are so large in proportion to the mother that they cannot pass through the pelvis as it normally exists. This is one of the strangest of those strange adaptations which make the reproductive functions of one species different from those of another, and which puzzle and confuse the investigator, but sometimes offer unexpected clues if he is sharp enough to see them.
Looking for the cause of this pelvic relaxation in the guinea pig, Hisaw and his young fellow students sought for a hormone ("relaxin") in the corpus luteum. In the course of this quest they obtained, and were the first to mention (1928), although without exact definition, extracts having some of the properties now known to be those of the corpus luteum hormone. The matter of the supposed relaxative hormone, incidentally, still remains a puzzle about which too little is known to discuss it in this book.
With one of these preparations Weichert (1928) was able to duplicate Leo Loeb's experiment (described above) in a castrated guinea pig; that is, the corpus luteum extract acted upon the guinea pig's uterus so that in response to irritation of its lining it produced masses of maternal cells as in pregnancy. This was confirmed by several of my students. Thus the basic functions of the corpus luteum suspected by Ancel and Bouin and by Loeb were confirmed by the use of extracts. Fraenkel's findings were also soon repeated with the hormone, for as soon as Willard Allen and I could prepare enough of the extract, we were able (1930) to castrate female rabbits at the eighteenth hour after mating and to carry the embryos, by use of our extract, all the way to normal birth at the usual term of pregnancy, in seven of our first fourteen attempts.
It should be added that this is a tricky experiment about which even yet we do not have full information. It seems to require just the right amount of estrogen along with the progesterone. Perhaps we were lucky that our extract was just sufficiently impure. At any rate it has turned out to be much harder to accomplish the same result with chemically pure progesterone.
Although the experiments thus far cited were all done upon the rabbit and guinea pig, there is no doubt that similar effects are produced in other species, including the human. In 1930 Hisaw and Fevold were able to produce progestational proliferation in the monkey, and the same result has since been achieved many times in women by gynecologists who had occasion to administer progesterone to human patients whose ovaries had been removed. There will be much more to say about this when we come to discuss the menstrual cycle in Chapter VI.
Meanwhile Willard Allen was makmg successful efforts to purify the hormone. Our crude extracts were already free of protein and we had got rid of the phosphorus-containing fats, always a bother in alcoholic extracts. The next stages were much harder than it was to purify estrone, because unfortunately the corpus luteum hormone is destroyed by alkalies. The chemists, therefore, could not get rid of fatty contaminants by merely turning them into soap and washing them away. By one trick and another, however, Allen successively got rid of the major contaminants, namely fats, fatty acids, and the inert sterol known as cholesterol. He began to get the hormone in crystalline form. Fels and Slotta, then (1931) in Breslau, Fevold and Hisaw (1932) and Allen (1932) ultimately obtained an almost perfectly pure crystalline substance of high potency. For his part of this work Willard Allen received the 1935 Eli Lilly Award of the American Chemical Society for the best work in biochemistry done by an American under thirty-one years of age. We now called into consultation Dr. Oskar Wintersteiner of Columbia University, a skilled microanalyst. Allen gave him 75 milligrams of the crystals, not much more than the weight of a postage stamp. He completed the purification and found that the hormone is a sterol having 21 atoms of carbon, 30 of hydrogen and 2 of oxygen. Practically at the same time Slotta, Ruschig and Fels at Breslau, and Butenandt and Westphal, then at Danzig, also reported ultimate purification of the hormone and announced, as probably correct, the formula printed below. Butenandt told me later that the total amount of the hormone he had available with which to work out its structure was 20 milligrams, one-third of the weight of a postage stamp. The same year (1934) Butenandt and two colleagues succeeded in making the hormone synthetically by chemical manipulation and rearrangement of a better known and more widely available sterol and thus confirmed the formula.
The chemistry of progesterone. This hormone is also a sterol, and is put together in a way not greatly unlike the estrogenic substances. Students of organic chemistry will recognize it as 3, 20 diketo 4, 5 pregnene :
A more detailed explanation of its chemical relationships will be found in Appendix I. This substance has not yet been synthesized from simple materials, but it has been made by rearranging the structure of somewhat more complicated sterols built up by plants and animals. For some years a vegetable sterol from soy beans was the most readily available source for the synthetic chemist, but it is now being made from cholesterol, which occurs plentifully in the spinal cord of oxen.
The name progesterone. It finally became necessary to name this substance, even before we knew what it was, in order to avoid long phrases in talking about it. Our experiments had proved that its effects are progestational, i.e. it favors gestation; for this reason I decided, with Willard Allen's approval, to call it progestin. This word is easy to spell and pronounce in many tongues, means something but not too much, and did not commit us to any theories that might prove untenable later. When the exact chemical nature of the hormone became known, the chemists, led by the amiable and diplomatic Butenandt, suggested the suffix -sterone. This tells us that the substance is a sterol containing doubly-linked oxygen.
Progesterone it is, then; and my original name progestin is retained for use as a general term when we need to talk about such a hormone without specifying its exact chemical structure. As a matter of fact, some of the other sterols have been found to produce progestational proliferation, though rather feebly in comparison with progesterone itself, and as a group we can call these "progestins," just as we use estrogen as a general term and specify estrone, estradiol, etc. as individual substances.
Natural sources of progesterone. The corpus luteum is the only important natural source of progesterone. It has been extracted not only from the sow, but also from the whale, whose 2-pound corpus luteum contains a large amount. A progestin (probably progesterone) has been extracted from the cow. There is a little in the human placenta, and most curiously of all, the adrenal gland contains something that gives similar effects in animals.
Potency of progesterone; administration. This hormone is not as potent, weight for weight, as the estrogens. Direct comparisons are scarcely possible, for the two kinds of substances do different things ; but if we compare the amounts necessary to produce definite effects in the whole uterus and whole vagina of an animal respectively, we have to use doses of progesterone several hundred times larger than of estrogen. Like coal and dynamite, they exert their power in different ways.
In 1935 the League of Nations Commission on Biological Standardization agreed upon an international unit of progesterone, namely the amount of potency in 1 milligram of the chemically pure hormone. This is 1/60 of the weight of a postage stamp. To compare an unknown preparation with this, it must be tested on rabbits under standard conditions.
Progesterone is soluble in oils and fats as well as in fat solvents like ether, and therefore it is generally injected hypodermically in a bland vegetable oil, such as sesame oil. It is ineffective when given by mouth, as shown by extensive trials on rabbits. Recently some of the drug manufacturers have put out another substance, a progestin of slightly different chemical structure, which is reported to give progestational proliferation in rabbits when given by mouth. Its usefulness in human patients is now being established (Appendix II, note 5).
A failure and what it taught. Willard Allen and I had a queer experience with our first extracts, from which we learned something important, so that the story is not only amusing but useful. The beginning of this tale is that when we started we followed (as I said before) a hint from the work of Edmund Herrmann, who had obviously produced progestational proliferation in a few of his experiments without knowing it. He had used very young rabbits, roughly 8 weeks old. They react more readily than adults to the estrogen which was the chief ingredient of his extracts. Since we wanted to follow his methods closely at first, we used infant rabbits too, and with them our first successes were obtained. In the spring of 1929 we were all ready to report the first steps in print. The paper was being written, when it occurred to me that our directions for extracting the hormone ought to be tried out by a nonetoo-good chemist, just to make sure they were foolproof. We did not want others to think our work could not be repeated, just because our directions were not clear. It was agreed that I was a bad enough chemist for the test : if I could make the extract all by myself, then anybody could. So Allen went on his vacation and I went back to our extractors and vacuum stills. In a week I had a batch ready; to my horror it was ineffectual. I made another batch; it, too, was worthless. I suppressed the paper and telegraphed for Allen. We decided that I needed a vacation and that we would look for the trouble in the fall. In September I made another batch with Allen watching every step, but not touching the apparatus. It was no good. What could be wrong. Since my laboratory was sunnier than his, perhaps my hormone was being spoiled by sunlight. I had a room blacked out and made a batch in the dark. That failed. Then we remembered that Allen, being a better chemist than I, usually got his extracts freer of superfluous grease and therefore had to mix them with corn oil (Mazola) so that he could inject them. Mine were greasy enough to inject without added oil. Perhaps the corn oil protected his hormones somehow while mine spoiled. We checked that idea â€” another two weeks gone â€” and that was not the answer. Then in desperation we made a batch together, side by side and almost hand in hand, each watching the other. We divided it into two lots and tested it separately - Allen's worked ; mine did not ! Eureka, my trouble was in the testing, not in the cookery.
The explanation will seem so silly that I almost hesitate to admit what it was. The fact is that rabbits do not respond well to progesterone until they are about 8 weeks old and weigh about 800 grams. We did not know this, and our rabbits ranged from 600 to 1,200 grams. When we went to the cages to inject them, Willard Allen's idea of what constitutes a nice rabbit led him to choose the larger ones, while I must have had a subconscious preference for the infants. My extracts had been as good as his all the while, but my rabbits were insensitive. It is staggering to think how often the success or failure of research may hang upon such an unimaginable contingency.
We thought very hard about this experience, and decided that after all we should not have expected that the hormone of gestation would act upon an infant's uterus. Caring for embryos is a job for a grown-up uterus. Thereafter we used only adult rabbits and never had another failure. Before we published the method one of our laboratory technicians made a lot of the extract unassisted and it worked.
Meanwhile the Wisconsin workers, Hisaw and his colleagues, had found that their extracts that relaxed the guinea pig's pelvic bones would not work in infant guinea pigs unless the animal was first "primed" with estrogenic hormone. Following up this clue, Allen learned that if we make the infant uterus grow larger by a few days' treatment with an estrogen, it will then respond fully to progesterone. A very striking experiment illustrating this is shown in Plate XVIII, E, An infant rabbit has, of course, a very small uterus. After 5 days' treatment with estrogenic hormone, the uterus becomes large and well differentiated. If progesterone is then given for 5 days, the uterus shows full progestational proliferation like that of an adult in early pregnancy. Meanwhile the rabbit herself remains a baby small enough to hold on the palm of one's hand.
By what means does progesterone affect the uterus? Just how the various hormones exert their action upon the organs which they control is very obscure. About the action of progesterone we have at present only a very remote idea. We can guess that in some way the hormone alters the general nutritional state of the cells of the uterus by way of a change in their metabolic rate or some other deep-seated effect on cellular physiology.
What happens to progesterone after it acts? In 1936 a discovery of great importance was made by Ethel H. Venning and J. S. V. Browne of Montreal. This is that in the human body used-up progesterone is converted into another substance called pregnanediol (preg-nane-di-ol). The conversion takes place by the addition of six atoms of hydrogen. I give the chemical formula for those who are interested:
Pregnanediol is an inert substance as far as hormone action is concerned. The body gets rid of it by attaching it to another substance readily available from the starches and sugars of the food, namely glycuronic acid (see Appendix, p. 253). To this an atom of sodium is added, and the combined substance, sodium pregnanediol glycuronidate, passes out through the kidneys. It happens to be easily separable from the urine and can thereafter be detected and measured by relatively simple laboratory tests. Each molecule of this waste product in the urine means that a molecule of progesterone was available for conversion. If ten milligrams, for instance, of pregnanediol is recoverable from a single day's urine, then we know the patient produced at least that much progesterone - in fact a little more, for there is some loss in the chemical process of measurement and possibly some loss in the body, i.e. some of the progesterone may be broken down and eliminated in other ways. The method is good enough, however, to help very much in estimating the functional activity of the corpus luteum in women and is beginning to be used as a method of diagnosing ovarian deficiencies.
All this information about the conversion and excretion of progesterone unfortunately holds good only for the human species and (apparently) the chimpanzee. It is enough to make a laboratory experimenter tear his hair, when he realizes that in other animals progesterone is excreted in some other way, which nobody has been able to discover. Pregnanediol has not been found in the urine of rabbits and monkeys, nor in any other animal. Nor has any other substance that might be derived from progesterone and from nothing else been found in the urine. Either it escapes from the body, in lower animals than man, in some elusive form ; or it is broken down so completely that its chemical fragments are undistinguishable among the simple remnants of bodily chemicals that make up the excretions (Appendix II, note 6).
Fig. 17. Arrangement of apparatus for maintaining excised pieces of uterine muscle under physiological conditions and recording its contractions.
Action of progesterone on the muscle of the uterus
In 1927 a well-known Austrian scientist, Hermann Knaus, discovered another action of the corpus luteum. To make this clear let us imagine an experiment such as is shown in Fig. 17. A rabbit is killed, the uterus is removed, and a portion of one horn is suspended in a cylinder of salt solution so that one end is tied down and the other is fastened to a writing lever that writes on a revolving drum. The salt solution is kept at body temperature by standing in a bath of warm water. Oxygen is bubbled through the salt solution, and a little sugar is put into it as food, providing energy for the tissues. The whole thickness of the uterus, except its inner lining, is composed of involuntary muscle, as explained on page 48. Placed in an apparatus of the sort just described, which imitates the natural conditions within the body, any such piece of living involuntary muscle, whether from the uterus, intestine, stomach, bladder, blood vessels, or elsewhere, will undergo rhythmic contractions every minute or two and will write them on the revolving drum as shown in the illustration. The experimenter, if he likes, can induce larger, more sudden contractions by putting into the salt solution one or another of those substances that are known to stimulate involuntary muscle. A drop of adrenalin solution will make the uterine muscle pull so strongly that it will almost yank the lever ofF the drum. So will a drop of pituitrin solution (subject to a very important reservation which is about to be mentioned) â€” indeed, pituitrin is such a powerful stimulant of uterine muscle that it is often used by obstetricians to make the human uterus contract after childbirth.
Knaus found that the uterus of a pregnant rabbit, set up in salt solution as described, will not react to pituitrin. He had just learned this when the first reports of the isolation of progestin reached Austria ; he made up some crude progestin (the first in Europe) and found that when he gave this by injection to a castrated adult rabbit for 5 days, the uterus became absolutely insensitive to pituitrin, exactly as if she were pregnant.
This experiment works with some species of animals and not with others ; we do not know surely about the human uterus in this respect. In cats, strangely enough, progesterone suppresses the action of adrenalin on the uterus (something that never happens in the rabbit) but does not suppress the action of pituitrin. These differences present a remarkable and probably very subtle problem in the physiology of muscle. When it is answered we shall know much more than we do now about involuntary muscle and also about hormones. Details aside, however, the experiment of Knaus shows us that progesterone can act in a striking way on involuntary muscle. We owe to S. R. M. Reynolds a very ingenious method of studying uterine contractions in living rabbits. By a simple plastic operation, done in a few minutes under complete anesthesia, the twin cervices of the uterus are stitched to the belly wall and thus made accessible. A rabbit so prepared suffers no inconvenience if properly cared for, and like any other healthy unfrightened rabbit will lie quietly on her back for hours if gently tied down. A tiny rubber balloon is carefully inserted inside the uterus. From this a tube leads to a little bellows which actuates a lever writing upon a revolving smoked drum. Whenever the uterus contracts, the balloon is squeezed, the bellows is inflated, and the lever goes up. With this apparatus Reynolds found that the normal uterus of an adult female rabbit undergoes more or less regular contractions. Castration suppresses the contractions, for the uterus which is enfeebled by castrate atrophy (see page 79) becomes inactive. Administration of estrogenic hormone, however, restores the contractions. Progesterone promptly and effectively quiets the uterus. The graph shown herewith (Fig. 18) illustrates the effect of an injection of progesterone. At 10:20 a.m. the uterus was contracting regularly. The hormone was given a few minuteis later. In one hour (see third line of the graph) the contractions became definitely smaller and less frequent.
By 12:20 p.m. the uterine muscle was practically not contracting at all. This effect wears off in a few hours.
Such a sedative action of progesterone upon the uterine muscle has been observed in many animals and there seems to be good evidence that it occurs in the human species. In all probability it serves to keep the uterus quiet in early pregnancy so that the embryos can become safely implanted.
Fig. 18. Effect of progesterone on the contractions of the living rabbit's uterus. A tiny rubber bulb in the cavity of the uterus is compressed each time the uterine muscle squeezes down, so that the rhythmic contractions are recorded on a revolving drum. Progesterone administered at 10:20 a.m. By 12:20 p.m. the uterus is quiescent. From an article by S. R. M. Reynolds and W. M. Allen, 1935, by courtesy of the American Journal of Obstetrics and Gynecology and the C. V. Mosby Company.
Is the corpus luteum necessary throughout pregnancy? In the rat and mouse, removal of the ovaries at any time in gestation causes termination of pregnancy. The embryos are cast off prematurely or they break down in the uterus and are absorbed in situ. Guinea pigs do not always lose their young if the ovaries are removed after implantation has occurred. In humans it seems certain that both ovaries, including of course the corpus luteum, can be removed after the first few weeks without harm. ^Vhether the pregnant human female, and other animals having long terms of pregnancy, do not need progesterone after the embryos are safely implanted, or whether perhaps the placenta makes enough to serve in place of the corpus luteum, we do not yet know (Appendix II, note 7).
Progesterone as a medicinal drug
Progesterone is already in the drugstores. It comes in neat little boxes of glass ampoules filled with a bright clear oily solution, duly labeled with the hormone content in international units. Tablets of the similar substance that acts by mouth (page 118) are also available for trial. Prescriptions for these drugs will be filled no less readily, though somewhat more expensively, than for digitalis or belladonna.
Before we expect the doctors to cure people with the ovarian hormones, however, let us consider the special circumstances. To take a quite different case, when Banting, Best, Collip, and McLeod handed over the pancreatic hormone, insulin, to the medical profession, they were filling a specific, clearly understood need. They had worked out the insulin problem by experiments on dogs. Sugar is sugar, whether a dog burns it or a man, and the way in which different animals use sugar is the same, regardless of the species. Diabetes, moreover, was a well understood disease, and in the minds of the medical profession it was waiting to be treated with this hormone as a lock waits for the key.
To take another example, the hormone of the adrenal medulla, epinephrin (trade name Adrenalin) has a relatively simple, direct action. What it will do, what it is needed for, is fairly clear. It can be used for acute asthma, or for bleeding from mucous membranes, with understanding and with reasonable hope that it will be effective under the circumstances.
This is unfortunately not the case with endocrine disease of the reproductive system. The human uterus and the rest of the system behave quite differently from those of most other animals. We cannot, for example, apply directly to humans with regard to disturbances of menstruation or pregnancy, information gained from the lower mammals, because the latter do not menstruate and in pregnancy they differ in many ways. It will be clear enough when we deal with the menstrual cycle, in Chapter VI, that we do not even yet fully understand the way the ovarian hormones take part in menstruation. We know that the monthly cycle and the process of gestation require not only the individual hormones but also an exact balance between them. When these things go wrong, they do so in complex and devious ways. When a young woman is cramped with menstrual pain or a wife goes childless against her will, in many cases neither her physician nor the investigator in his laboratory can say exactly what is wrong or how to redress it. They can only try their best ; often the treatment works, often not. Only by the slow pathway of experiments on monkeys and cautious observation and trial directly in humans shall we ever comprehend the normal physiology of reproduction in our own species and those vexatious, oftentimes tragic disturbances that lead to disorders of menstruation, miscarriage, and sterility.
It is therefore only in small degree possible as yet to apply progesterone and estrone, and the other potent steroidal hormones, to human disease. We must leave the problem in the hands of competent gynecologists and obstetricians, particularly in the clinics of the medical schools and research hospitals. When these men give the word, hormone treatment becomes justifiable. The work is going forward daily; I do not mean to be discouraging, but only to avoid false promises of quick magic like that of insulin. Incidentally some cases of diabetes still defy insulin, and the specialists in that disease have by no means been able to declare their treatment perfected and their researches complete.
The maintenance of pregnancy
What can we hope for from progesterone in the long run ? The great dramatic thing about this hormone as seen in the laboratory is of course its power to maintain pregnancy after the loss of ovarian function. One of the greatest problems of medical practice is that of spontaneous abortion of the embryo or fetus ("miscarriage"). About one pregnancy in three terminates prematurely, according to generally accepted figures. These accidents have many causes. Sometimes the embryo is itself unhealthy, through some mischance of heredity or development, like a seedling plant that will not grow. Sometimes illness or accident to the mother upsets the course of events. Sometimes, we may suppose, the hormones go wrong. Perhaps the supply of progesterone from the corpus luteum or placenta is not adequate. In such cases it might conceivably be useful to supply the hormone by injection, thus making up the lack. The main difficulty at present is to diagnose such cases in time to treat them.
There is another way in which progesterone might help. No matter what the cause of an abortion, it is always accompanied by spasmodic contractions of the uterus, trying to get rid of its burden. Sometimes, we think, the contractions come first, as the result of injury or illness, and dislodge a normal embryo. We have seen that progesterone will quiet the uterine muscle; by reason of this, we may hope that it will help to steady the uterus in the case of a threatening abortion. With these thoughts in mind, the doctor tries progesterone in the face of such a disaster. Sometimes the pregnancy goes on, sometimes not. How can he tell whether his hormone worked the cure ? His case records are not altogether helpful, for no two cases are alike; and what is more, the physician in his anxiety generally tries two or three remedial measures at once, any one of which might have been responsible. If he controls a large clinic with many such patients, he can use the treatment on alternate cases only until he is satisfied which group does better. By this method, while the physician is being scientific he is (if the drug is really useful) condemning half his patients with threatened abortion to the risk of losing their babies - a dilemma similar to that dramatically expounded in Sinclair Lewis's great novel of medical life, Arrowsmith. A clever method of testing this question was recently suggested by the British physicians Malpas, McGregor and Stewart, who pointed out that women who are unfortunate enough to have three or more successive spontaneous abortions are (statistically speaking) almost certain to miscarry in the next subsequent pregnancy. If, then, any kind of treatment is followed by the birth of a living infant, the odds are great that the medical procedure, and not mere chance, was responsible. By this severely critical test, it appears that progesterone is saving some of these babies. Needless to say, a treatment which is still so largely experimental requires skilled and thoughtful handling, by physicians thoroughly familiar with the proper dosage and other problems.
Post partum pain
The most clear-cut use of progesterone involving its sedative action on the uterine muscle is for the relief of spasmodic pain due to excessive contractions of the uterus after childbirth. This is sometimes severe enough to require relief. Lubin and Clarke, of Brooklyn, found that a single dose of one international unit of progesterone will relieve these afterpains in about 90 per cent of the cases.
Painful menstruation is one of the commonest of human ills, and one of the least understood. Knowing as little as we do about normal menstruation, it is no wonder that we also know all too little about its disturbances. In the case of painful menstruation (dysmenorrhea) we are not even sure of the exact seat of the pain in all cases. It is probably due to cramping of the muscular wall of the uterus ; but there is reason to think that in some cases the pain may be produced in the lining of the uterus rather than in the muscular substance of the wall. All sorts of treatment have been tried, from psychological analysis to operations designed to correct faulty positions of the uterus. The fact that each of these widely different measures sometimes succeeds and sometimes fails, suggests strongly that dysmenorrhea is not a single disease, but rather a symptom due to different causes in different cases. The whole situation creates a problem for investigation by the combined forces of clinical gynecology and the laboratory investigators. Unfortunately, we can expect to get very little help, in such a problem as this, from study of animals. Even the female Rhesus monkey, so useful for study of the physical aspects of menstruation, cannot help us in this investigation, for even if she suffered from dysmenorrhea or could be made to experience uterine cramps for the purpose of our studies, she cannot report her symptoms or help us evaluate the results of treatment.
When it was discovered that progesterone can quiet the normal contractions of an animal's uterus and even the violent spasms that cause post partum pain in human patients, many physicians thought of trying it in dysmenorrhea, thinking that it might relieve a crampy state of the uterus. As usual whenever a new treatment, no matter what, is tried in one of the old reliable guaranteed-to-baffle diseases, some of the doctors and patients reported hopeful results. A little later skeptical reports began to be published. Critical observers reminded us anew that dysmenorrhea is such a peculiar thing that we must be very cautious about accepting a new cure. For example, there are undoubtedly some cases in which the pain is largely subjective, arising from psychic causes. These people will be helped by any treatment that happens to win their confidence. Depending upon the patient's turn of mind, a hypodermic injection of sterile water given with due assurance, any new hormone in an impressive package, psychological or religious comfort â€” any of these may give genuine relief. With equal certainty there are other cases produced by some sort of actual physical or chemical disorder in the reproductive system, and these must be attacked, if possible, by treatment aimed directly at the cause. The physician, however, cannot definitely classify these cases before he treats them. Being a merciful man and anxious to give relief as soon as possible, he generally prescribes what has worked best in his last few cases. He usually tries several things at once, thus spoiling a good experiment in the hope of more relief. As a result, it is very difficult to judge the effects of progesterone when used in the treatment of dysmenorrhea. What is badly needed is a large-scale report from one of the university hospital clinics, based on a long series of cases in which progesterone has been used in alternate patients, and in alternate periods in the same patient, so that really scientific checkup of the effects can be provided. Meanwhile, there have been a good many reports of relief of menstrual cramps, some of them almost magical, and other reports of failure. It certainly ought to be tried in cases that have resisted other forms of treatment, and that are severe enough to warrant the necessary hypodermic injections, as well as the expense, which may run up to several dollars at each period if large doses are necessary. If the new progesterone-like drugs for administration by mouth prove to be successful, they will simplify the problem.
Control of irregular or excessive menstrual bleeding
Progesterone has the property of preventing menstruation, as we shall see when we discuss that subject in Chapter VIII. For this reason it is being tried in cases of excessive menstrual bleeding and irregularity. It looks as if this hormone and some of its chemical relatives are going to be really useful in these distressing ailments, as we come to understand them better ; but this is decidedly a matter for trained specialists. No drug can safely be used to stop uterine bleeding except after a thorough examination, to rule out the possibility of bleeding from cancer or other tumor of the uterus. Once such causes of bleeding as these are ruled out, the physician may safely try progesterone.
At the present time each case of menstrual disorder is a separate problem and both doctor and patient must realize that hormone treatment is experimental. It brings relief from debilitation and misery to some women even now. As we learn more through cautious trial, more will be helped. Meanwhile those of us who have had something to do with the finding of this hormone wish to see it exploited with care and understanding, not discredited by premature advertising and incautious use (Appendix II, note 8).
- Photographs of the rabbit's uterus magnified, shown in this book, represent sections (slices) across the uterus, as one slices a banana.
- Now Profepsor of Obstetrics and Gynecology, Washington University, and head of the St, Louis Maternity Hospital.
- This statement need cause no alarm to any prospective mother who happens to read it. Once pregnancy is past the first weeks and under medical care, it goes safely on in an overwhelming majority of cases. The figure of one loss in three includes many pregnancies of the earliest weeks, and even some that occur so early as to be recognized only by microscopic methods.
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